升麻三萜皂苷对破骨细胞形成分化的影响及机制研究  

Effect and mechanism of Cimicifuga triterpene saponins on formation and differentiation of osteoclast

作　　者：宋子乐 张奇 胡思婧 张泉龙 徐金龙 邱明华[3] 张巧艳 韩婷 秦路平 SONG Zile;ZHANG Qi;HU Sijing;ZHANG Quanlong;XU Jinlong;QIU Minghua;ZHANG Qiaoyan;HAN Ting;QIN Luping(School of Pharmaceutical Sciences,Zhejiang Chinese Medical University,Hangzhou 310053,China;969 Hospital of PLA Joint Logistics Support Forces,Hohhot 010051,China;Kunming Institute of Botany,Chinese Academy of Science,Kunming 650201,China;School of Pharmacy,Naval Medical University,Shanghai 200433,China)

机构地区：[1]浙江中医药大学药学院,浙江杭州310053 [2]中国人民解放军联勤保障部队第九六九医院,内蒙古呼和浩特010051 [3]中国科学院昆明植物研究所,云南昆明650201 [4]中国人民解放军海军军医大学药学院,上海200433

出　　处：《中草药》2024年第12期4032-4044,共13页Chinese Traditional and Herbal Drugs

基　　金：国家自然科学基金面上项目(82374098);上海市科委科技支撑计划项目(22S21901600)。

摘　　要：目的探究升麻三萜皂苷(Cimicifuga triterpene saponins)对破骨细胞形成分化和骨吸收功能的影响,并基于网络药理学探讨其作用机制。方法采用CCK-8法测定升麻三萜皂苷阿克特素、升麻环氧醇苷、升麻醇对骨髓巨噬细胞(bone marrow macrophages,BMMs)增殖活性的影响;抗酒石酸酸性磷酸酶(tartrateresistant acid phosphatase,TRAP)染色测定破骨细胞数目;磷酸苯二钠法测定破骨细胞的TRAP活性;鬼笔环肽染色观察破骨细胞F-actin环的形成;甲苯胺蓝染色检测破骨细胞在牛皮质骨骨片上形成的骨吸收陷窝;Western blotting分析破骨细胞标志基因和蛋白的表达。采用GeneCards和DisGeNET数据库获取破骨细胞功能的靶点,应用Cytoscape 3.7.1软件,采用蛋白互作的方式,筛选升麻三萜皂苷调控破骨细胞功能的核心靶点,并采用DAVID 6.8对核心靶点进行基因本体(gene ontology,GO)功能及京都基因与基因组百科全书(Kyoto encyclopedia of genes and genomes,KEGG)通路富集分析。结果升麻三萜皂苷对BMMs增殖无显著影响,升麻三萜皂苷阿克特素、升麻环氧醇苷、升麻醇均可减少破骨细胞的数目,抑制破骨细胞TRAP活性和F-actin环的形成,显著抑制破骨细胞活化T细胞核因子1(nuclear factor of activated T cells 1,NFATc1)、原癌蛋白Fos(oncogene Fos,c-Fos)、基质金属蛋白酶9(matrix metalloproteinase 9,MMP9)和组织蛋白酶(cathepsin K,CTSK)的表达(P<0.05、0.01),减少破骨细胞在骨片上形成的骨吸收陷窝的面积(P<0.01)。网络药理学分析表明升麻三萜皂苷主要通过丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)通路调控破骨细胞的骨吸收功能。结论升麻三萜皂苷通过MAPK通路抑制BMMs诱导的破骨细胞的形成分化和骨吸收,可为抗骨质疏松新药的研发提供先导化合物。Objective To explore the effects of Cimicifuga triterpene saponins on osteoclast(OC)formation,differentiation and bone resorption,and explore the mechanism based on network pharmacology.Methods The effects of C.triterpene saponins actein,cimigenoside and cimigenol on proliferation of bone marrow macrophages(BMMs)were determined by CCK-8 assay;The number of OC was measured by tartrateresistant acid phosphatase(TRAP)staining;The TRAP activity of OC was determined by phenylene disodium phosphate method;The formation of F-actin ring of OC was observed by phalloidin staining;The formation of bone resorption lacunae of OC in bovine cortical bone slices was detected by toluidine blue staining;The expressions of OC marker gene and protein were detected by Western blotting.The targets involved into the regulation of osteoclast function were obtained through GeneCards and DisGeNET database,and the core targets of C.triterpene saponins in regulation of osteoclast function were screened by Cytoscape 3.7.1 software and protein-protein interaction.DAVID 6.8 was used to perform gene ontology(GO)function and Kyoto encyclopedia of genes and genomes(KEGG)pathway enrichment analysis of core targets.Results C.triterpene saponins had no significant effect on the proliferation of BMMs.C.triterpene saponins actein,cimigenoside and cimigenol reduced the number of OC,inhibited the activity of TRAP and the formation of F-actin ring of OC,and significantly decreased the expressions of nuclear factor of activated T cells 1(NFATc1),oncogene Fos(c-Fos),matrix metalloproteinase 9(MMP9)and cathepsin K(CTSK)(P<0.05,0.01),and decreased the area of bone absorption lacunae on bone slices of OC(P<0.01).Network pharmacology analysis showed that C.triterpene saponins regulated the bone resorption function of OC mainly through mitogen-activated protein kinase(MAPK)pathway.Conclusion C.triterpene saponins inhibited formation,differentiation and bone resorption activities of OC derived from BMMs through MAPK pathway,which provides lead compounds for the

关 键 词：升麻 三萜皂苷 阿克特素 升麻环氧醇苷 升麻醇 破骨细胞 网络药理学 丝裂原活化蛋白激酶通路 

分 类 号：R285.5[医药卫生—中药学]